Sea buckthorn (Hippophae rhamnoides) is an ecologically and economically valuable species that has been widely cultivated as a new berry crop rich in nutritional and medicinal compounds. RNA Sequencing (RNA-Seq) simple sequence repeat (SSR) markers were developed to evaluate the genetic relationships among 91 plants of 31 cultivars from two subspecies, mongolica and sinensis, as well as intraspecific hybrids between them. A total of 7540 RNA-Seq SSRs were identified as potential molecular markers, in which AG/CT (27.57%) was the most abundant unit type. AT/AT (9.93%), and AAG/CTT (11.95%) are the other main repeat motifs. A total of 110 primer pairs were randomly selected for validation of amplification. Seventeen SSR loci, located in genes encoding metabolic processes and cellulose synthases, were identified to be polymorphic among different sea buckthorn cultivars. These SSR loci generated 48 alleles, ranging from 2 to 5 per locus. Cluster analysis based on the proportion of shared alleles and unweighted pair group method with arithmetic average (UPGMA) algorithm divided all the genotypes into two main groups, with all of the ssp. sinensis cultivars (native to China) and hybrids in one group and ssp. mongolica cultivars (introduced from Russia) in the other group, which was in good agreement with their taxonomic classification. The RNA-Seq SSRs developed in this study have a potential use in the conservation of sea buckthorn germplasm and marker-assisted breeding (MAB).